Cardiac muscle regeneration using mesenchymal stem cells

ABSTRACT

Disclosed is a method for repairing or regenerating blood vessels in the heart of an individual, method of stimulating or promoting angiogenesis in the heart of an individual, or method of stimulating or promoting vascular endothelial growth factor (VEGF) expression in a heart of an individual by administering to the individual an effective amount of mesenchymal stem cells. These cells can be administered as a liquid injectable.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/012,953,filed on Jan. 25, 2011, which is a continuation of application Ser. No.12/395,949, filed on Mar. 2, 2009 (now U.S. Pat. No. 7,892,829, issuedFeb. 22, 2011), which is a continuation of application Ser. No.10/690,435, filed Oct. 21, 2003 (now U.S. Pat. No. 7,514,074, issuedApr. 7, 2009), which is a continuation-in-part of application Ser. No.10/278,148, filed Oct. 22, 2002, now abandoned, which is acontinuation-in-part of application Ser. No. 10/127,737, filed Apr. 22,2002 (now abandoned), which is a continuation of application Ser. No.09/446,952, filed Mar. 27, 2000 (now U.S. Pat. No. 6,387,369, issued May14, 2002), which is the national phase application of PCT ApplicationNo. PCT/US98/14520, filed Jul. 14, 1998, which claims priority of U.S.provisional application Ser. No. 60/052,910, filed Jul. 14, 1997, thecontents of which are incorporated by reference in their entireties.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[Not Applicable]

BACKGROUND OF THE INVENTION

This invention relates to the replacement and regeneration of cardiactissue and muscle.

This year over 300,000 Americans will die from congestive heart failure.The ability to augment weakened cardiac muscle would be a major advancein the treatment of cardiomyopathy and heart failure. Despite advancesin the medical therapy of heart failure, the mortality due to thisdisorder remains high, where most patients die within one to five yearsafter diagnosis.

A common heart ailment in the aging population is improper heart valvefunction, particularly the aortic valve. Mechanical replacement valvesare widely used but require the patient to continually take bloodthinners. Valves obtained from cadavers and xenographs (porcine) arealso frequently used to replace a patient's own tissue. Valves arefreeze-dried or chemically cross-linked using e.g., glutaraldehyde tostabilize the collagen fibrils and decrease antigenicity and proteolyticdegradation. However, these valves remain acellular and often fail afterseveral years due to mechanical strain or calcification. A replacementvalve derived from biocompatible material that would allow ingrowth ofthe appropriate host cells and renewal of tissue over time would bepreferred.

Mesenchymal stem cells (MSCs) are cells which are capable ofdifferentiating into more than one type of mesenchymal cell lineage.Mesenchymal stem cells (MSCs) have been identified and cultured fromavian and mammalian species including mouse, rat, rabbit, dog and human(See Caplan, 1991, Caplan et al. 1993 and U.S. Pat. No. 5,486,359).Isolation, purification and culture expansion of hMSCs is described indetail therein.

SUMMARY OF THE INVENTION

In accordance with the present invention mesenchymal stem cells (MSCs)are used to regenerate or repair striated cardiac muscle that has beendamaged through disease or degeneration. The MSCs differentiate intocardiac muscle cells and integrate with the healthy tissue of therecipient to replace the function of the dead or damaged cells, therebyregenerating the cardiac muscle as a whole. Cardiac muscle does notnormally have reparative potential. The MSCs are used, for example, incardiac muscle regeneration for a number of principal indications: (i)ischemic heart implantations, (ii) therapy for congestive heart failurepatients, (iii) prevention of further disease for patients undergoingcoronary artery bypass graft, (iv) conductive tissue regeneration, (v)vessel smooth muscle regeneration and (vi) valve regeneration. Thus theMSCs are also used to integrate with tissue of a replacement heart valveto be placed into a recipient. The MSCs, preferably autologous,repopulate the valve tissue, enabling proper valve function.

MSC cardiac muscle therapy is based, for example, on the followingsequence: harvest of MSC-containing tissue, isolation/expansion of MSCs,implantation into the damaged heart (with or without a stabilizingmatrix and biochemical manipulation), and in situ formation ofmyocardium. This approach is different from traditional tissueengineering, in which the tissues are grown ex vivo and implanted intheir final differentiated form. Biological, bioelectrical and/orbiomechanical triggers from the host environment may be sufficient, orunder certain circumstances, may be augmented as part of the therapeuticregimen to establish a fully integrated and functional tissue.

Accordingly, one aspect of the present invention provides a method forproducing cardiomyocytes in an individual in need thereof whichcomprises administering to said individual a myocardium-producing amountof mesenchymal stem cells. The mesenchymal stem cells that are employedmay be a homogeneous composition or may be a mixed cell populationenriched in MSCs. Homogeneous human mesenchymal stem cell compositionsare obtained by culturing adherent marrow or periosteal cells; themesenchymal stem cells may be identified by specific cell surfacemarkers which are identified with unique monoclonal antibodies. A methodfor obtaining a cell population enriched in mesenchymal stem cells isdescribed, for example, in U.S. Pat. No. 5,486,359. Compositions havinggreater than about 95%, usually greater than about 98%, of humanmesenchymal stem cells can be achieved using techniques for isolation,purification, and culture expansion of mesenchymal stem cells. Forexample, isolated, cultured mesenchymal stem cells may comprise a singlephenotypic population (about 95% or about 98% homogeneous) by flowcytometric analysis of expressed surface antigens. The desired cells insuch composition are identified as expressing a cell surface marker, forexample, CD73, CD105, or CD166. These cell surface markers arespecifically bound by an antibody produced from hybridoma cell line SH2,ATCC accession number HB 10743; an antibody produced from hybridoma cellline SH3, ATCC accession number HB 10744; or an antibody produced fromhybridoma cell line SH4, ATCC accession number HB 10745.

The administration of the cells can be directed to the heart, by avariety of procedures. Localized administration is preferred. Themesenchymal stem cells can be from a spectrum of sources including, inorder of preference: autologous, allogeneic, or xenogeneic. There areseveral embodiments to this aspect, including the following.

In one embodiment of this aspect, the MSCs are administered as a cellsuspension in a pharmaceutically acceptable liquid medium for injection.Injection, in this embodiment, can be local, i.e. directly into thedamaged portion of the myocardium, or systemic. Here, again, localizedadministration is preferred.

In another embodiment of this aspect, the MSCs are administered in abiocompatible medium which is, or becomes in situ at the site ofmyocardial damage, a semi-solid or solid matrix. For example, the matrixmay be (i) an injectible liquid which “sets up” (or polymerizes) to asemi-solid gel at the site of the damaged myocardium, such as collagenand its derivatives, polylactic acid or polyglycolic acid, or (ii) oneor more layers of a flexible, solid matrix that is implanted in itsfinal form, such as impregnated fibrous matrices. The matrix can be, forexample, Gelfoam (Upjohn, Kalamazoo, Mich.). The matrix holds the MSCsin place at the site of injury, i.e. serves the function of“scaffolding”. This, in turn, enhances the opportunity for theadministered MSCs to proliferate, differentiate and eventually becomefully developed cardiomyocytes. As a result of their localization in themyocardial environment they then integrate with the recipient'ssurrounding myocardium. These events likewise occur in the above liquidinjectible embodiment, but this embodiment may be preferred where morerigorous therapy is indicated.

In another embodiment of this aspect, the MSCs are genetically modifiedor engineered to contain genes which express proteins of importance forthe differentiation and/or maintenance of striated muscle cells.Examples include growth factors (TGF-β, IGF-1, FGF), myogenic factors(myoD, myogenin, Myf5, MRF), transcription factors (GATA-4), cytokines(cardiotrophin-1), members of the neuregulin family (neuregulin 1, 2 and3) and homeobox genes (Csx, tinman, NKx family). Also contemplated aregenes that code for factors that stimulate angiogenesis andrevascularization (e.g. vascular endothelial growth factor (VEGF)). Anyof the known methods for introducing DNA are suitable, howeverelectroporation, retroviral vectors and adeno-associated virus (AAV)vectors are currently preferred.

Thus, in association with the embodiment of the above aspect usinggenetically engineered MSCs, this invention also provides novelgenetically engineered mesenchymal stem cells and tissue compositions totreat the above indications. The compositions can include geneticallymodified MSCs and unmodified MSCs in various proportions to regulate theamount of expressed exogenous material in relationship to the totalnumber of MSCs to be affected.

The invention also relates to the potential of MSCs to differentiatepartially to the cardiomyocyte phenotype using in vitro methods. Thistechnique can under certain circumstances optimize conversion of MSCs tothe cardiac lineage by predisposing them thereto. This also has thepotential to shorten the time required for complete differentiation oncethe cells have been administered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show cardiac muscle injected, using a fine needle, with invitro dye-labeled MSCs. The lipophilic dyes PKH26 (Sigma Chemical) orCM-Di I (Molecular Probes) were utilized to label MSCs prior to beingintroduced into animals. These dyes remain visible when the tissue siteis harvested 1-2 months later. We have also shown that such dyes do notinterfere with the differentiation of MSCs in in vitro assays. FIG. 1Ashows the low magnification image of a rat heart which has been injectedwith dye labeled cells and later, a T-incision has been made at thesite. FIGS. 1A and 1B reveal the labeled MSCs in the ventricle wallviewed from the outer surface. FIG. 1C shows a cross-section of theventricle wall and that the cells are present in the outer 1-2 mm of the3 mm thick cardiac muscle.

FIG. 2. Comparison of MSC engraftment when delivered to rats via directcardiac injection (Panel A) or tail vein (Panel B). Confocal images wereobtained in hearts harvested 4 weeks post-implantation.

FIG. 3 shows images indicative of anterior wall motion in infarctedswine hearts that received no treatment and those that were treated withallogeneic MSCs.

FIG. 4 shows graphs of ejection fraction (upper panels) measured ininfarcted swine hearts that received no treatment and those that weretreated with MSCs, and graphs of global wall motion (lower panels) ininfarcted swine hearts that received no treatment, and those that weretreated with MSCs.

FIG. 5 is a graph of end diastolic pressure in infarcted swine heartsthat received no treatment and those that were treated with MSCs.

FIGS. 6A and 6B show sections of an infarcted region of a pig heart at 8weeks after being treated with DAPI-labeled mesenchymal stem cells. Bothfigures show the presence of blood vessels in the infarcted region. FIG.6A is a hematoxylin and eosin stained section, while FIG. 6B is afluorescent image showing the mesenchymal stem cells (dark, or blue) andof smooth muscle actin (light, or green), wherein the section wascontacted with an FITC-labeled monoclonal antibody against smooth muscleactin.

FIGS. 7A through 7E show sections of an infarcted pig heart at 12 weeksafter being treated with DAPI-labeled mesenchymal stem cells. Thefigures show the presence of blood vessels in the infarcted region.FIGS. 7A and 7D are hematoxylin and eosin stained sections. FIG. 7B is afluorescent image of DAPI-labeled mesenchymal stem cells. FIG. 7C is afluorescent image showing the presence of DAPI-labeled mesenchymal stemcells (dark, or blue) and of Factor VIII (light, or green), wherein thesection was contacted with an FITC labeled monoclonal antibody againstFactor VIII. FIG. 7E is a fluorescent image showing the presence ofDAPI-labeled mesenchymal stem cells (dark, or blue), and of vascularendothelial growth factor (VEGF), wherein the section was contacted withan FITC-labeled monoclonal antibody against VEGF.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The proper environmental stimuli convert MSCs into cardiac myocytes.Differentiation of mesenchymal stem cells to the cardiac lineage iscontrolled by factors present in the cardiac environment. Exposure ofMSCs to a simulated cardiac environment directs these cells to cardiacdifferentiation as detected by expression of specific cardiac musclelineage markers. Local chemical, electrical and mechanical environmentalinfluences alter pluripotent MSCs and convert the cells grafted into theheart into the cardiac lineage.

Early in embryonic development following the epithelia-mesenchymetransition, the presumptive heart mesenchyme from the left and rightsides of the body migrate to the ventral midline. Here, interaction withother cell types induces continued cardiogenesis. In vitro conversion ofMSCs to cardiomyocytes is tested by co-culture or fusion with murineembryonic stem cells or cardiomyocytes, treatment of MSCs with cardiaccell lysates, incubation with specific soluble growth factors, orexposure of MSCs to mechanical stimuli and electrical stimulation.

A series of specific treatments applicable to MSCs to induce expressionof cardiac specific genes are disclosed herein. The conditions areeffective on rat, canine and human MSCs. Treatments of MSCs include (1)co-culturing MSCs with fetal, neonatal and adult rat cardiac cells, (2)use of chemical fusigens (e.g., polyethylene glycol or sendai virus) tocreate heterokaryons of MSCs with fetal, neonatal and adultcardiomyocytes, (3) incubating MSCs with extracts of mammalian hearts,including the extracellular matrix and related molecules found in hearttissue, (4) treatment of MSCs with growth factors and differentiatingagents, (5) mechanical and/or electrical stimulation of MSCs, and (6)mechanically and/or electrically coupling MSCs with cardiomyocytes. MSCsthat progress towards cardiomyocytes first express proteins found infetal cardiac tissue and then proceed to adult forms. Detection ofexpression of cardiomyocyte specific proteins is achieved usingantibodies to, for example, myosin heavy chain monoclonal antibody MF 20(MF20), sarcoplasmic reticulum calcium ATPase (SERCA1) (mAb 10D1) or gapjunctions using antibodies to connexin 43.

Cardiac injury promotes tissue responses which enhance myogenesis usingimplanted MSCs. Thus, MSCs are introduced to the infarct zone to reducethe degree of scar formation and to augment ventricular function. Newmuscle is thereby created within an infarcted myocardial segment. MSCsare directly infiltrated into the zone of infarcted tissue. Theintegration and subsequent differentiation of these cells ischaracterized, as described above. Timing of intervention is designed tomimic the clinical setting where patients with acute myocardialinfarction would first come to medical attention, receive first-linetherapy, followed by stabilization, and then intervention withmyocardial replacement therapy if necessary.

Of the four chambers of the heart, the left ventricle is primarilyresponsible for pumping blood under pressure through the body'scirculatory system. It has the thickest myocardial walls and is the mostfrequent site of myocardial injury resulting from congestive heartfailure. The degree of advance or severity of the congestive heartfailure ranges from those cases where heart transplantation is indicatedas soon as a suitable donor organ becomes available to those wherelittle or no permanent injury is observed and treatment is primarilyprophylactic.

The severity of resulting myocardial infarction, i.e. the percentage ofmuscle mass of the left ventricle that is involved can range from about5 to about 40 percent. This represents affected tissue areas, whether asone contiguous ischemia or the sum of smaller ischemic lesions, havinghorizontal affected areas from about 2 cm² to about 6 cm² and athickness of from 1-2 mm to 1-1.5 cm. The severity of the infarction issignificantly affected by which vessel(s) is involved and how much timehas passed before treatment intervention is begun.

The mesenchymal stem cells used in accordance with the invention are, inorder of preference, autologous, allogeneic or xenogeneic, and thechoice can largely depend on the urgency of the need for treatment. Apatient presenting an imminently life threatening condition may bemaintained on a heart/lung machine while sufficient numbers ofautologous MSCs are cultured or initial treatment can be provided usingother than autologous MSCs.

The MSC therapy of the invention can be provided by several routes ofadministration, including the following. First, intracardiac muscleinjection, which avoids the need for an open surgical procedure, can beused where the MSCs are in an injectible liquid suspension preparationor where they are in a biocompatible medium which is injectible inliquid form and becomes semi-solid at the site of damaged myocardium. Aconventional intracardiac syringe or a controllable arthroscopicdelivery device can be used so long as the needle lumen or bore is ofsufficient diameter (e.g. 30 gauge or larger) that shear forces will notdamage the MSCs. The injectible liquid suspension MSC preparations canalso be administered intravenously, either by continuous drip or as abolus. During open surgical procedures, involving direct physical accessto the heart, all of the described forms of MSC delivery preparationsare available options.

As a representative example of a dose range is a volume of at leastabout 20 μl, preferably at least 500 μl, of injectible suspensioncontaining 10−40×10⁶ MSCs/ml. The concentration of cells per unitvolume, whether the carrier medium is liquid or solid remains withinsubstantially the same range. The amount of MSCs delivered will usuallybe greater when a solid, “patch” type application is made during an openprocedure, but follow-up therapy by injection will be as describedabove. The frequency and duration of therapy will, however, varydepending on the degree (percentage) of tissue involvement, as alreadydescribed (e.g. 5-40% left ventricular mass).

In cases having in the 5-10% range of tissue involvement, it is possibleto treat with as little as a single administration of one million MSCsin 20-50 μl of injection preparation. The injection medium can be anypharmaceutically acceptable isotonic liquid. Examples include phosphatebuffered saline (PBS), culture media such as DMEM (preferablyserum-free), physiological saline or 5% dextrose in water (D5W).

In cases having more in a range around the 20% tissue involvementseverity level, multiple injections of 20-50 μl (10−40×10⁶ MSCs/ml) areenvisioned. Follow-up therapy may involve additional dosings.

In very severe cases, e.g. in a range around the 40% tissue involvementseverity level, multiple equivalent doses for a more extended durationwith long term (up to several months) maintenance dose aftercare maywell be indicated.

When given intravenously, the mesenchymal stem cells may be administeredin at least 20 μl, preferably at least 500 μl, and up to about 150 ml ofa suspension containing 10−40×10⁶ MSCs/ml. In one embodiment, from 40 mlto about 150 ml of a suspension containing 10−40×10⁶ MSCs/ml is givenintravenously.

Applicants also have discovered that the mesenchymal stem cells maystimulate and/or promote angiogenesis in the heart and/or repair orregenerate blood vessels of the heart. Thus, in accordance with anotheraspect of the present invention, there is provided a method ofstimulating or promoting angiogenesis in the heart, or of repairing orregenerating blood vessels of the heart of an individual byadministering to the individual mesenchymal stem cells in an amounteffective to stimulate or promote angiogenesis, or repair or regenerateblood vessels of the heart. The mesenchymal stem cells may beadministered as a cell suspension in a pharmaceutically acceptableliquid medium such as hereinabove described, or in a biocompatiblemedium which is, or becomes in situ at the site of myocardial damage, asemi-solid or solid matrix, also as hereinabove described.

The mesenchymal stem cells may be allogeneic, autologous, or xenogeneic,and may be administered in dosages such as those hereinabove described.

When the mesenchymal stem cells are administered as a cell suspension ina pharmaceutically acceptable liquid medium for injection, they may beadministered locally, i.e., directly into the damaged portion of theheart, such as by an endocardial catheter for example, or they may beadministered systemically, such as by intravenous administration.

The mesenchymal stem cells provide for the repair or regeneration ofexisting blood vessels of the heart, as well as promote angiogenesis,i.e., the formation of new blood vessels of the heart. Blood vesselswhich may be repaired or regenerated, as well as new blood vessels whichmay be formed, include arteries (including arterioles), and veins, aswell as capillaries.

The present invention is further illustrated, but not limited, by thefollowing examples.

EXAMPLE 1 Implantation of MSCs in Normal Cardiac Muscle

In using MSCs, it is desirable to maintain cell-cell contact in vivo forthe conversion of MSCs to the muscle lineage. Environmental signalsidentified above act in concert with mechanical and electrical signalingin vivo to lead to cardiac differentiation.

Primary human MSCs (hMSCs) are introduced into athymic rat myocardialtissue by direct injection. The integration of implanted cells, theirsubsequent differentiation, formation of junctions with cardiac cells,and their long-term survival are characterized with light microscopy,histology, confocal immunofluorescence microscopy, electron microscopyand in situ hybridization.

Whether human MSCs are appropriately grafted into cardiac muscle ofathymic rats (strain HSD:RH-RNU/RNU), which lack the immune responsesnecessary to destroy many foreign cells, is also examined.

Rat MSCs are grafted into the heart muscles of rats. To analyze theinjected cells over several weeks and to minimize the possibility ofimmune system rejection, MSCs are harvested from Fisher 344 rats, thesame inbred strain (identical genotype) as the intended MSC recipients.

The MSCs can be marked in a variety of ways prior to their introductioninto the recipient. This makes it possible to trace the fate of the MSCsas they proliferate and differentiate in the weeks following the MSCimplant. Several methods are utilized to identify positively theinjected cells: membrane lipid dyes PKH26 or CM-DI I and genetic markingwith adeno-associated virus (AAV) or retroviruses, such as Moloneymurine leukemia virus expressing green fluorescent protein (GFP) orgalactosidase. PCR also is used to detect the Y chromosome marker ofmale cells implanted into female animals. The dye-labeled cells aredetected readily and offer the simplest method to directly follow theinjected cells. This method is reliable for times out to at least 4weeks. On the day of introduction to recipient animals, MSCs aretrypsinized and labeled with CM-DI I according to the recommendations ofthe manufacturer (Molecular Probes). Subconfluent monolayer cultures ofMSCs are incubated with 5 mM CM-DI I in serum-free medium for 20minutes, trypsinized, washed twice in excess dye-free medium, andutilized for injection.

Alternatively, MSCs are genetically marked prior to injections, such asby using AAV-GFP vector. This vector lacks a selectable marker butmediates high-level expression of the transduced genes in a variety ofpost-mitotic and stem cell types. Recombinant AAV-GFP is added to lowdensity monolayers of MSCs in low serum. Following a four hourincubation at 37° C., the supernatant is removed and replaced with freshmedia. At 96 hours after transduction, cells are assayed for greenfluorescent protein (GFP) activity. Typically 50% of the cells expressthe transduced gene. Unselected MSCs on a clonal line, isolated bylimiting dilution, are utilized for injection. Cells are collectedfollowing trypsin treatment, washed and used at high concentrations forinjection (10 to 100 million cells per ml).

To test whether the hMSCs became cardiomyocytes in the heartenvironment, the hearts of ten week old athymic rats were injected withdye labeled or GFP-labeled human MSCs. All procedures were performedunder strict sterile conditions. The animals were placed in a glass jarcontaining a methoxyflurane anesthesia soaked sponge. Under sterileconditions, a 20 mm anterior thoracotomy was performed, and followingvisualization of the left ventricle, 10 μl of the cell suspension,containing 10,000 to 100,000 MSCs in serum-free medium were injectedinto the left ventricular apex using a 30 gauge needle. The procedurewas performed rapidly with endotracheal intubation and mechanicalventilation assist. The incision was closed with sutures. Ventilationassist was normally unnecessary after a short period following chestclosure. FIG. 1A shows the low magnification image of a rat heart whichwas injected with dye labeled cells and later, a T-incision had beenmade at the site to reveal the injected cells in the ventricle wall.FIG. 1A is a gross photo of the incised heart. FIGS. 1B and 1C revealthe labeled MSCs in the ventricle wall. FIG. 1C shows that the cellswere present in the outer 1-2 mm of the 3 mm thick rat cardiac muscle.

When sacrificed, the heart is removed, examined by light microscopy forthe presence of vascular thrombi or emboli, paraffin-embedded, andsectioned. The histology of serial sections is examined to determine thefate of dye-stained cells. Sections then are tested forimmunohistochemical markers of cardiac muscle in the areas of theintroduced MSCs to ascertain whether donor MSCs have differentiated intocardiomyocytes in vivo. Implantation surgeries are carried out onanimals to be sacrificed at 1, 2, 4, and 6 weeks (4 animals at each timepoint) and the hearts which received implants are analyzedhistologically and immunologically.

For phenotypic characterization, the hearts are removed and processedfor histology by immunofluorescence microscopy. Differentiation of MSCsis determined by the immunofluorescence localization of sacomeric myosinheavy chain, SERCA1 and phospholamban. The sequence-specific antibody togap junction protein connexin 43, which is commercially available(Zymed) and detects gap junctions in cardiac tissue is used.

MSCs are also implanted in biomatrix materials to determine if enhancedgrafting would be observed, such as Type I collagen. The MSCs are mixedrapidly with the matrix in a small volume and injected into theventricle wall. The biomatrices are used at concentrations of 0.1 mg/mlor greater. For example, the biomatrices may be used at concentrationsof 1 to 3 mg/ml containing 10 to 100 million cells/ml. The tissue isanalyzed at times of 1, 2, 4, and 6 weeks as described above.

EXAMPLE 2 Regeneration of Heart Valves Using MSCs

Xenograft or homograft valves are made acellular by freeze-drying, whichleads to cellular death, or by enzymatic treatment followed by detergentextraction of cells and cell debris. This latter approach was taken byVesely and coworkers with porcine valves to be repopulated with dermalor aortic fibroblasts. Curtil, et al. 1997 used a freeze-dried porcinevalve and attempted repopulation of the valve with human fibroblasts andendothelial cells. These studies were preliminary and limited to shortterm studies in vitro.

The acellular valve to be populated by autologous hMSCs is incubatedwith culture expanded hMSCs in a tumbling vessel to ensure loading ofcells to all valve surfaces. The valve is then cultured with the hMSCsfor 1-2 weeks to allow the hMSCs to infiltrate and repopulate the valve.Within the culture vessel, the valve is then attached to a pump to allowthe actuation of the valve leaflets and simulate the pumping motionpresent in the body. The valve is maintained in the pumping mode for 1-2weeks to allow cellular remodeling associated with the stresses of thepumping action. Once sufficient cellular remodeling has occurred, thevalve is implanted into the body of the patient.

Another embodiment of this aspect of the invention is to firstrepopulate the valve with hMSCs and to later incubate the valve tissueduring the pumping stage with autologous smooth muscle cells isolatedfrom a vascular graft which will line the lumen of the valve.

EXAMPLE 3 MSC Engraftment in Rat MI Model: Direct Injection vs. SystemicDelivery

Myocardial infarction was produced in Fisher rats as follows:

Fisher rats were given a cocktail of Ketamine/Xylazine/Acepromazine (8.5mg/1.7 mg/0.3 mg I.P.) The depth of anesthesia was assessed using atoe-pinch and eye-blink reflexes. When a surgical plane of anesthesiawas achieved, endotracheal intubation was performed and the animalplaced on 1.0% Isoflorane. Positive-pressure breathing was providedthroughout the procedure by means of the Engler ADS 1000 small animalventilator. A left thoracotomy was performed and the pericardium opened.A 6-0 silk ligature snare was then placed around the left anteriordescending (LAD) coronary artery at a location distal to the firstdiagonal branch. A brief (30 sec) LAD test occlusion is performed toinsure that a modest region of ischemia is procued, involving a limitedregion of the anterior free wall and septum. Ischemia is confirmed bycharacteristic ECG changes, ventricular dyskinesis and regionalcyanosis. Myocardial infarction is then produced by occluding the LADfor a period of 45 minutes. At the completion of the 45 minute period,the snare is removed and reperfusion visually confirmed. The chest wasthen closed by approximating the ribs and all associated musculature.The Isoflurane is turned off, the animal removed from the ventilator andextubated.

Panel A of FIG. 2 shows engraftment of MSCs in the heart followingdirect injection into the heart. In these experiments, 2−4×10⁶allogeneic rat MSCs were implanted into the area of necrosis by directinjection.

Panel B of FIG. 2 shows that tail vein injection results in cardiacengraftment.

These animals received MSCs via the tail vein. Injection of theallogeneic cell suspension occurred when the animals had stabilized, anda normal cardiac rhythm had been reestablished; usually within 15minutes of reperfusion. At that time approximately 5×10⁶ MSCs in a 0.5milliliter suspension were injected slowly into the tail vein.

EXAMPLE 4

Swine are sedated with 1000 mg ketamine IM and brought into the lab.Intravenous access is established via an ear vein and the animalsanesthetized with nembutal. Swine then are intubated, ventilated with1.0-1.5% isoflurane, and prepped for surgery. ECG leads and rectaltemperature probes are placed and the animal is draped to create asterile field. A midline sternotomy is performed and the heart suspendedin a pericardial cradle. A tygon catheter is placed in the apex of theleft ventricle and sutured in place to measure ventricular pressurethroughout the cardiac cycle. The left anterior descending (LAD)coronary artery is dissected free just distal to the first diagonalbranch. A brief (30 sec) occlusion of the coronary artery is performedto identify the regions of ischemia (identified by the extent ofcyanosis). Four piezoelectric crystals then are placed within regionsdestined for infarction.

At the completion of the surgical instrumentation a 15 minutestabilization period is allowed prior to obtaining baseline recordings.Following these recordings, the LAD there is occluded for a period of 60minutes to produce myocardial infarction. Lidocaine (local anestheticand antiarrhythmic) is administered at this time to reduce the incidenceof ventricular fibrillation (2 mg/kg i.v. bolus plus 0.5 mg/min ivdrip). Recordings of left ventricular pressure and regional contractilefunction are again obtained at 10 and 50 minutes of ischemia. Extensivecyanosis within the ischemic bed was noticed following 50 minutes ofischemia.

At the completion of the 60 minute period of ischemia, the snare isreleased and reperfusion established. Care is taken to ensure thatperfusion is reestablished and that the isolated region of the LAD isnot in spasm. At this time the leads (sono leads and LV catheter) areexternalized, and the chest closed in layers. A chest tube is placed toreestablish a negative intrapleural pressure (tube is pulled 24 hrslater). The isoflurane is then turned off, and the animal is extubatedand allowed to recover.

One set of infarcted swine was treated with allogeneic mesenchymal stemcells and another set (control) did not receive such treatment. Theanimals were examined using echocardiography. In the mesenchymal stemcell treatment, a 10 ml MSC suspension was drawn up into several 3 ccsyringes using an 18 g needle. The needle was switched to a 30 g fordelivery. Implantation was accomplished in the open chest setting. Theneedle was advanced to the mid-wall level, and 0.5 mls of cells wereinjected. This same procedure was performed approximately 20 timesthroughout the damaged area. Care was taken to provide cells to theentire apical anterior wall, as well as the septum. At the completion ofthe implantation procedure, the chest was closed and the animal allowedto recover.

FIG. 3 contains “m-mode” images obtained in a control and an MSC treatedanimal. The image illustrates wall motion in a selected plane over time(moving left to right). The infarcted region of myocardium, consistingprimarily of anterior LV free wall, is the structure highlighted by thearrows. That segment of myocardium is essentially akinetic in thecontrol image, indicative of severe infarction/injury. While notquantifiable, there is improved anterior wall motion in the animaltreated with allogeneic MSCs.

Echocardiography was used to measure the ejection fraction, a measure ofglobal pump efficiency (a normal ejection fraction of 70% indicates that70% of the LV volume is pumped with each beat of the heart; EF<40% isindicative of heart failure). Ejection fraction data is shown in theupper panels of FIG. 4. Control animals demonstrated no significantimprovement in EF over the course of the study. In contrast, astatistically significant improvement in cardiac pump function wasobserved in MSC treated animals (right panel).

A similar graph was used to represent wall motion score index (lowerpanels of FIG. 4). In this analysis, 17 segments of the left ventriclewere examined for wall motion and scored on a scale of 1-5, with 1representing “normal” wall motion. These segments, comprising all areasof the ventricle, can then be averaged to gather an index of global wallmotion (i.e., global function). As with ejection fraction, nosignificant improvement in wall motion was observed in control animalsover time. MSC treated animals showed consistent and significantimprovements in wall motion scores over time (right panel).

Further evidence for improved cardiac function with MSC treatment isfound when end diastolic pressure (EDP) is examined. When cardiac pumpfunction is reduced following infarction, a pathologic increase in leftventricular EDP is observed. This increase in EDP is a clinicallyrelevant finding that is often predictive of the progression to heartfailure following infarction. As shown in FIG. 5, the EDP of controlswine rose from approximately 12 to 35 mmHg in the 6 months followinginfarction. The rise in EDP in animals treated with MSCs wassignificantly attenuated at all time points examined post-infarction.

EXAMPLE 5

Pathologic ventricular remodeling following myocardial infarction is amajor cause of heart failure. It was previously demonstrated thatautologous mesenchymal stem cells (MSC) augment local systolic wallthickening and prevent pathologic wall thinning. Based on in vitrostudies, it was hypothesized that MSCs may be immuno-privileged, andthat implantation of allogeneic MSCs could prevent pathologic remodelingand improve cardiac performance in a swine model of myocardialinfarction. Piezoelectric crystals and an LV catheter were implanted indomestic swine prior to a 60′ LAD occlusion to produce infarction.Following reperfusion, treated animals (n=7) were injected withallogeneic Dil-labeled MSCs (2×10⁸ cells in 9 ml) throughout the regionof infarction. Control (CON, n=6) received vehicle. Allogeneic donorMSCs were previously isolated from swine iliac crest bone marrow,expanded in culture, and cryopreserved until the time of implantation.Hemodynamic parameters and regional wall motion were evaluated inconscious animals bi-weekly using trans-thoracic echocardiography andsonomicrometry. Animals were sacrificed at various time points (6-24weeks) and tissue harvested for histological examination. Implantationof allogeneic MSCs was not associated with ectopic tissue formation,significant inflammatory response or any adverse clinical event. Robustengraftment of allogeneic MSCs was observed in all treated animals.Furthermore, engrafted MSCs were found to express numerous musclespecific proteins, and exhibited morphological changes consistent withmyogenesis. A marked improvement in both ejection fraction (55±9.4% vs32.5±12.5% in CON) and global wall motion score (1.45±0.15 vs 2.1±0.2 inCON) was observed in treated animals at 10 weeks post-MSC implantation.Systolic wall thickening and diastolic wall thickness were alsoaugmented in MSC treated animals. Because no significant difference ininfarct size or cardiac loading was noted between groups, improvementsin cardiac function are likely attributable to MSC implantation. Inconclusion, this example suggests that implantation of allogeneic MSCsat reperfusion may be an effective therapeutic option to prevent orreverse the progression to heart failure following infarction.

The above examples illustrate that mesenchymal stem cells augmentventricular function, as shown, for example by improved cardiac ejectionfraction and global wall motion.

EXAMPLE 6

A pig was subjected to a 60 minute LAD occlusion to produce infarctionas described in Example 5. Three days after the infarction, 200×10⁶diaminopropidium iodide (DAPI)-labeled allogeneic mesenchymal stem cellswere administered to the left venticular wall by endocardial catheter as20 separate injections of 10×10⁶ cells each in 0.5 ml physiologicalsaline. DAPI is a nuclear stain which emits a strong blue fluorescenceand aids in the identification of implanted cells.

Eight weeks after administration of the mesenchymal stem cells, the pigwas sacrificed, and the heart was harvested for histologicalexamination. Sections were subjected to hematoxylin and eosin staining,or to fluorescence imaging after being contacted with an FITC-labeledmonoclonal antibody against smooth muscle actin.

The hematoxylin and eosin image (FIG. 6A) clearly illustrates thepresence of blood vessels within a generalized region of myocardialnecrosis.

As shown in FIG. 6B, DAPI-labeled cells can be seen throughout thesection; however, a localization of implanted MSC's can be identifiedreadily. These MSC's surround, and are associated with, the bloodvessels.

As shown in FIG. 6B, the lighter, or green, fluorescence indicates thepresence of the FITC-labeled monoclonal antibody against smooth muscleactin, thus indicating the presence of a blood vessel. Also present inFIG. 6B are DAPI-labeled (blue) MSC's localized within such vessel, andwhich are associated intimately with the smooth muscle layer of thevessel. Thus, the MSCs are involved in the repair or regeneration ofblood vessels of the heart.

EXAMPLE 7

A pig was subjected to a 60 minute LAD occlusion to produce infarctionas described in Example 5. Three days after the infarction; the pig wasgiven 200×10⁶ diaminopropidium iodide (DAPI)-labeled allogeneicmesenchymal stem cells as 20 separate injections of 10×10⁶ cells in 0.5ml physiological saline into the left ventricular wall by endocardialcatheter as described in Example 6.

Twelve weeks after administration of the mesenchymal stem cells, the pigwas sacrificed, and the heart was harvested for histologicalexamination. Sections were subjected to hematoxylin and eosin staining,or to fluorescence imaging after being contacted with an FITC-labeledmonoclonal antibody against Factor VIII (Von Willebrand Factor) or anFITC-labeled monoclonal antibody against vascular endothelial growthfactor (VEGF).

As shown in the hematoxylin and eosin images of FIGS. 7A and 7D, thepresence of blood vessels within a region of generalized myocardialnecrosis is illustrated.

In the fluorescent images of FIGS. 7B, 7C and 7E, DAPI labeled cells canbe seen throughout the sections; however, localizations of MSCs can beidentified which surround and are associated intimately with the smoothmuscle layer of the blood vessels. Light, or green, fluorescenceindicates the presence of FITC-labeled monoclonal antibody againstFactor VIII (FIG. 7C) or against VEGF (FIG. 7E).

Thus it has been shown that the implanted MSCs express Factor VIII andVEGF, which are indicative of angiogenesis. These proteins are notexpressed by cultured MSCs, but are expressed only after several weeksin the cardiac environment.

The disclosure of all patents and publications (including publishedpatent applications) are hereby incorporated by reference to the sameeffect as if each patent and publication were individually andspecifically incorporated by reference.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

1. A method of repairing or regenerating blood vessels of the heart ofan individual, comprising: administering to the individual allogeneicmesenchymal stem cells in an amount effective to repair or regenerateblood vessels in the heart of the individual, wherein the mesenchymalstem cells express one or more cell surface markers selected from thegroup consisting of CD73, CD105, and CD166.
 2. The method of claim 1,wherein the mesenchymal stem cells are capable of binding to an antibodyselected from the group consisting of an antibody produced by thehybridoma cell line SH2, ATCC accession number HB 10743, an antibodyproduced from the hydridoma cell line SH3, ATCC accession number HB10744, and an antibody produced from hydridoma cell line SH4, ATCCaccession number HB
 10745. 3. The method of claim 1, wherein themesenchymal stem cells are administered directly to the heart.
 4. Themethod of claim 1, wherein the mesenchymal stem cells are administeredin a pharmaceutically acceptable liquid injectable carrier.
 5. Themethod of claim 1, wherein the cardiomyocyte producing amount ofmesenchymal stem cells is administered by a catheter.
 6. The method ofclaim 1, wherein the cardiomyocyte producing amount of mesenchymal stemcells is administered in a matrix.
 7. A method of stimulating orpromoting angiogenesis in the heart of an individual, comprising:administering to the individual allogeneic mesenchymal stem cells in anamount effective to stimulate or promote angiogenesis in the heart ofthe individual, wherein the mesenchymal stem cells express one or morecell surface markers selected from the group consisting of CD73, CD105,and CD166.
 8. The method of claim 7, wherein the mesenchymal stem cellsare capable of binding to an antibody selected from the group consistingof an antibody produced by the hybridoma cell line SH2, ATCC accessionnumber HB 10743, an antibody produced from the hydridoma cell line SH3,ATCC accession number HB 10744, and an antibody produced from hydridomacell line SH4, ATCC accession number HB
 10745. 9. The method of claim 7,wherein the mesenchymal stem cells are administered directly to theheart.
 10. The method of claim 9, wherein the mesenchymal stem cells areadministered by intracardiac injection.
 11. The method of claim 7,wherein the mesenchymal stem cells are administered in apharmaceutically acceptable liquid injectable carrier.
 12. The method ofclaim 7, wherein the mesenchymal stem cells are administered by acatheter.
 13. The method of claim 7, wherein the mesenchymal stem cellsare administered in a matrix.
 14. A method of stimulating or promotingvascular endothelial growth factor (VEGF) expression in a heart of anindividual, comprising: administering to the individual allogeneicmesenchymal stem cells in an amount effective to stimulate or promoteVEGF expression in the heart of the individual, wherein the mesenchymalstem cells express one or more cell surface markers selected from thegroup consisting of CD73, CD105, and CD166.
 15. The method of claim 14,wherein the mesenchymal stem cells are capable of binding to an antibodyselected from the group consisting of an antibody produced by thehybridoma cell line SH2, ATCC accession number HB 10743, an antibodyproduced from the hydridoma cell line SH3, ATCC accession number HB10744, and an antibody produced from hydridoma cell line SH4, ATCCaccession number HB
 10745. 16. The method of claim 14, wherein themesenchymal stem cells are administered directly to the heart.
 17. Themethod of claim 14, wherein the mesenchymal stem cells are administeredin a pharmaceutically acceptable liquid carrier.
 18. The method of claim14, wherein the mesenchymal stem cells are administered intravenously.19. The process of claim 14, wherein the mesenchymal stem cells areadministered in a matrix.
 20. The process of claim 14, wherein themesenchymal stem cells are administered by a catheter.